Prolan

PROLAN (Prolan), one of the hormones of the anterior lobe of the pituitary gland, as well as an organ preparation containing the designated hormone. The name was given by B. Zondek (B. Zondek, 1926). In his research, the author managed to show that the hormone, by the nature of its specific action, can be viewed as consisting of two substances: Prolan A—the factor for follicle maturation and the production of the female sex hormone, and Prolan B—the factor for luteinization (formation of corpora lutea) of the follicles, without increasing the size and number of the latter. For now, commercial preparations containing both substances (A and B) together are available. Prolan is contained in: 1) the anterior lobe of the pituitary gland of humans and animals (adults, young, and fetuses) (on average containing 100-200 units of substance A and 10-50 units of substance B), 2) the decidua graviditatis of the first 4 months of pregnancy, 3) the corpora lutea graviditatis, 4) the placenta, starting from the 2nd month of pregnancy, 5) the mucous membrane of the fallopian tubes in intrauterine and ectopic pregnancy from the 2nd month, 6) the blood of pregnant women from the 2nd month of pregnancy (in non-pregnant women, prolan is absent in the blood), 7) in a few cases, prolan was found in umbilical cord blood, 8) in human milk in the first 2 days of the postpartum period, 9) the urine of pregnant women from the 35th day after the last menses, with 1 liter of urine in the first 8 weeks containing about 3,000-5,000 units (see below), in the 3rd-7th months about 3,000-6,000 units, and in the 7th-10th months 2,000-3,000 units; during pregnancy, both substances (A and B) are present in the urine. In non-pregnant healthy women, there is much less prolan in the urine, with the most during menstruation and the least (1 unit per 1 liter) in the intermenstrual period. Only substance A is excreted in the urine upon failure of ovarian function (in 25% of climacteric cases, after castration), in patients with severe endocrine diseases, tumors, and in very severe inflammatory processes. In men (both healthy and sick), no prolan was detected in the urine. It is assumed that the site of hormone production is the anterior lobe of the hypophysis, and during pregnancy also the placenta. The increase of prolan in urine and blood during pregnancy is explained either by the cessation of ovarian function, which normally inhibits prolan production, or by the absence of a site of application for prolan's action, since follicle formation ceases during pregnancy. The cheapest and most accessible method of obtaining prolan is according to Zondek's method from the urine of pregnant women, where the hormone is in a soluble form. However, prolan has not yet been obtained in a chemically pure form. Prolan isolated from urine is a fine, yellowish-white amorphous powder, very easily soluble in water, insoluble in lipoid solvents (alcohol, ether, acetone, benzene, chloroform, etc.). Prolan is very sensitive to temperature and begins to change already when heated to 60°, and is destroyed upon boiling. Passing oxygen through an aqueous solution of prolan for an hour does not destroy it. Prolan is very easily adsorbed, capable of dialysis, and is rapidly destroyed in strong acids and alkalis. It is not very sensitive to alcohol, so the addition of 8% alcohol does not change the activity of prolan for a week. Commercial liquid prolan preparations contain no protein or biogenic amines. Some authors indicate that the activity of prolan preparations does not change for several months. In 1922, Evans and Long (Evans, Long) showed that the injection of an extract from the anterior lobe of the hypophysis into the abdominal cavity of females affects the course of cyclical changes in the genital apparatus. Then they additionally reported that in young females, prolonged administration of the extract causes excessive formation of corpora lutea from follicles. In 1926, Teel showed that the extract causes the phenomenon of estrus in immature rats. In the same year, B. Zondek and Aschheim and independently of them Smith also obtained estrus in immature mice after transplantation of the anterior lobe to them and proposed this as a method (test) for detecting the hormone and quantifying its activity. Especially striking is the action of prolan on sexually immature mice, rats, and rabbits; the action of prolan on the latter is especially convincing because they lack spontaneous ovulation (it occurs only after coitus). After subcutaneous administration of fractional doses of prolan to rabbits, the following is observed: the thin and pale uterine horns turn into formations the thickness of a finger, of a bluish-red color. The uterine mucosa is succulent, thickened; vessels are hyperemic. The ovaries are enlarged 6-8 times, pinkish-red in color, their surface is uneven, dotted with dark blood spots, which are framed by golden-yellow corpora lutea. Sections through the vagina reveal an extremely sharp increase in muscle and connective tissue, and the mucosa presents the characteristic phenomena of estrus. In the uterus, the changes are even more pronounced: the mucosa grows massively, becomes polypoid, numerous mitoses are present in the epithelium, vessels are heavily engorged with blood, and there is much secretion in the cavity. In the ovary, small primordial follicles disappear, i.e., they are pushed out to the marginal zone, the ovary is filled with hyperemic and luteinized large follicles with cumulus oophorus; the egg is not expelled from the follicle, but is surrounded like a shell by yellow bodies (corpora lutea atretica). The cells of the stratum granulosum folliculi ovarii and especially the theca folliculi grow strongly and are luteinized. There are hemorrhages in the enlarged follicles. In old mice, in which ovarian function has long since ceased and there are no periodic phenomena of estrus, prolan causes follicle maturation and the formation of corpora lutea, so that the animals once again undergo the sexual cycle. Smith in many cases was able to eliminate prolan atrophy of the sex organs. In pregnant animals, prolan can force the ovary to function despite the presence of fetuses and placenta in the uterus—eggs mature and enter the fallopian tubes of the pregnant animal. If a large dose of prolan is given, the fetuses die in the uterus, abortion or maceration of the fetuses in the uterus occurs. In chickens, as Clark, Walker and others have shown, short-term injection of prolan increases egg-laying, while prolonged administration stops it. In pigeons, Riddle noted a 20-fold increase in testicles under the influence of 10-day prolan injections. In macaque monkeys, menstrual bleeding was successfully induced by prolan injection. In mammalian animals, prolonged administration of large doses of prolan also completely stops ovulation; hormonal sterilization in the sense of Haberlandt occurs. In order to convince oneself ad oculos of the action of small doses of prolan on women, Zondek injected prolan subcutaneously or intramuscularly for several days to patients who were to undergo abdominal surgery. After cutting the abdominal wall, he discovered marked hyperemia of the genital organs, turgescence and loosening of the tubes and uterus, colored blue-red, i.e., the picture of early pregnancy. In individual cases, "premature development of follicles containing the female sex hormone (proven by biological assay) and the formation of corpora lutea were observed, while the vaginal mucosa was in the interval stage with incipient secretion and glycogen deposition, i.e., the mucosa in its structure did not correspond to the state of maturity of the corpus luteum. As in animals, individual women showed individual variations in the intensity of the reaction. The temperature in the rectum and vagina rose (by 0.5° above normal) due to hyperemia of the pelvic organs. Uterine bleeding of a congestive rather than menstrual character was also observed. After castration, the described changes cannot be obtained from prolan in animals. It follows that prolan acts on the tubes, uterus, and vagina not directly, but secondarily—forcing the ovary to intensively produce the female sex hormone. Judging by experimental data, prolan does not possess a stimulating action on the anterior lobe of the hypophysis. Zondek considers the hormone of the anterior lobe of the hypophysis to be the engine of the gonadal function, and, as we shall see below, prolan manifests the same action in the male organism as well. Substance A is responsible for the enlargement of the follicles of the uterus and vaginal mucosa—inducing typical phenomena of estrus; substance B turns granulosa cells into lutein cells, causes the formation of corpora lutea, but does not lead to an increase in follicles and does not change the state of the uterus and vagina.—Prolan causes the appearance of milk in the mammary glands in immature females. In individual cases, it caused secretion from the mammary glands in women in the pre-climacteric period. Prolan affects the male sex glands and secondarily (due to the production of the male sex hormone) the sex organs of males. In an immature ram, Zondek and Aschheim, as well as de Jongh on rats and mice, obtained an enlargement of the sex organs (penis, prostate gland, and especially seminal vesicles) in the absence or negligible enlargement of the testicles. The maximum enlargement of the testicles was 50% compared to control animals, and of the seminal vesicles by 600%. The epithelium of the seminiferous tubules becomes multilayered, mature spermatozoa appear, secondary sexual characteristics appear, libido sexualis increases, and Leydig cells increase very strongly in number and size. On castrated animals, the described action

PROLAN

Effects on secondary sex characteristics and libido sexualis are not produced by prolan. It is believed that substance A has no effect on the male genital apparatus and that this action is characteristic only of substance B. Thus, prolan affects the gonads of both sexes, as a result of which de Jongh figuratively calls it the motor of the gonads ("Motor fur die Gonade") instead of Zondek's earlier definition of this hormone as the motor of the ovary ("Motor fur das Ovarium"). Prolan has no effect on the growth of animals. The action of prolan occurs upon subcutaneous and intramuscular administration, but not upon its administration per os (with the exception of rats). It is believed that prolan is destroyed in the gastrointestinal tract. Experiments on various species of animals have shown that, in general, the greater the weight of the animal, the more sensitive it is to prolan. Thus, if the minimum effective dose of prolan for a mouse weighing 6 g is taken as a unit (consequently per 1 g of weight there will be ¹/₆ of it), then for a rat weighing 30 g the minimum effective dose will be equal not to 5, but only to ¹/₆-¹/₁₅ of a unit (per 1 g of weight ¹/₁₈₀-¹/₄₅₀), and for a rabbit weighing 1,200 g not 200, but only 5 (per 1 g of weight ¹/₂₄₀). These ratios are of considerable interest in developing doses suitable for humans, since prolan can both induce ovulation and stop it. There are no chemical methods for determining the activity of prolan-type preparations. At Zondek's suggestion, activity is measured biologically on infantile (immature) mice or rats (the latter react less correctly) by finding the smallest dose that causes typical phenomena in the genital apparatus. The smallest amount of the preparation that, divided into 6 portions and injected over 36 hours, produces follicle maturation and a secondary estrus reaction (so-called reaction I) in an infantile mouse (weighing 6-8 g), respectively a rat, 100 hours after the first injection is taken as 1 mouse, respectively rat, unit of activity of substance A. For substance B, the unit of activity is considered to be the smallest amount of the preparation which, under the same conditions of administration, produces the formation of blood spots (reaction II) and the transformation of follicles into a corpus luteum (reaction III). Reaction I is not specific for prolan, since it is also produced by the female sex hormone. The described reactions are established macroscopically, and in doubtful cases under a microscope on a series of sections from ovaries fixed in Zenker's fluid and then stained. The activity of commercially available preparations is expressed in units of action, usually in mouse units (M. U.). 1 M. U. is approximately equal to 6-8 rat units. Before being released for sale, prolan-type preparations must be tested for their specific activity on infantile mice or rats. The content of activity units must be indicated on the label of the preparation. The activity of commercially available preparations varies (30-60 mouse units in 1 cm³). Foreign prolan contains 30-60 rat units in 1 cm³. Aqueous solutions of prolan very quickly lose their activity, as a result of which prolan preparations available on the market are often unfit for use. In addition, there are tablets of 30-150 rat units each, given for weeks and months. As a single dose for humans, Zondek proposes 2 cm³ of prolan (60 rat units). Other authors used 60 rat units (1 ampoule) 3 times a day, for 3-6 weeks in a row (Köhler). Dosage for various indications for the use of prolan has not yet been finally established. Prolan itself is apparently of low toxicity for humans, but since it cannot be sterilized by boiling (it is destroyed), some factories, for greater reliability of its sterility, add fairly large amounts of antiseptics to the preparation, for example, tricresol, which causes sharp phenomena at the injection site (redness, swelling, pain) and even general phenomena (elevated temperature, headache, general malaise, etc.). Prolan is indicated in cases of menstrual anomalies when they are due to hypofunction of the ovaries. Conversely, hyperhormonal types of amenorrhea are a contraindication for the use of prolan. It is reported that it has been possible to obtain a favorable effect from prolan in underdeveloped genital organs, in cases of adiposogenital dystrophy, in some cases of pituitary cachexia (in combination with insulin and other hormonal preparations). In the climacteric period, Zondek recommends using prolan only in that stage when ovarian function has already ceased completely. A positive result of treatment is obtained only from repeated administration of prolan, while its single administration does not give a lasting effect. There are reports of the use of large doses of prolan in humans (400-2,000 mouse units) to obtain hormonal sterilization (infertility), but experimental research (Mandelshtam and Chaykovsky) shows that large doses are dangerous because they cause degenerative changes in the ovaries in animals. In humans (12 cases by Martin), it was possible to obtain phenomena of ovarian function loss, which disappeared after the cessation of prolan administration. Zondek showed that the onset of pregnancy is immediately accompanied by the abundant excretion of prolan (specifically B) in the urine. Therefore, testing urine for prolan content can serve as a reaction (test) for the diagnosis of early pregnancy and ectopic pregnancy. The reaction succeeds only in humans and monkeys, but not in other animals. In humans, pregnancy can be diagnosed as early as 35 days after the last menstruation. Error was noted in a very small number of cases (1-2%). To perform the reaction, 2 portions (25-30 cm³ each) of morning urine (which contains more prolan) are taken, 1 drop of tricresol is added to each, if the urine is alkaline it is acidified with acetic acid, then filtered and injected into the peritoneal cavity of mice for 48 hours in 5 doses (0.2, 0.25, 0.3, 0.3, and 0.4 cm³). 100 hours after the 1st injection, the mouse is killed and its ovaries are examined as indicated above. Substances toxic to mice contained in the urine can be removed with ether, in which prolan is insoluble.